The long term objectives of this grant proposal are to investigate the mechanisms and effect of pregnancy on glucose insulin sensitivity in women with normal glucose tolerance (NGT) and gestational diabetes (GDM). The specific aims of this proposal are to: 1) determine the relationship between the changes in glucose insulin sensitivity and level of expression of post receptor insulin signaling intermediates in skeletal muscle in lean and obese women with NGT and GDM in late gestation and postpartum, 2) to determine the role of tumor necrosis factor alpha (TNFalpha) modulation of insulin receptor beta (IRbeta) and insulin receptor substrate I (IRS-1) in skeletal muscle in late pregnancy and 3) to characterize the effect of pregnancy on glucose metabolism, i.e. insulin sensitivity and pancreatic beta cell response postpartum in women with NGT and GDM. Specific aim #1 will evaluate longitudinally 12 women with NGT (6 lean and 6 obese) matched with 12 women with GDM (6 lean and 6 obese) at 30-36 weeks and 16 weeks postpartum. All subjects will be evaluated using the euglycemic clamp with infusion of [66/2/H2] glucose and have Bergstrom needle skeletal muscle biopsies. The specific methodology will allow us to estimate insulin sensitivity using Western blotting to measure changes in protein expression and competitive PCR to compare differences in mRNA expression during and after pregnancy. Specific aim #2 will evaluate 16 women at the time of elective cesarean delivery (8 NGT) and gynecologic surgery (8 CTL). All subjects will have densitometry and rectus muscle biopsies at the time of surgery. Skeletal muscle will be used to immunoprecipitate IRbeta and IRS-1. IRS-1 from pregnant NGT will be incubated with IRbeta from non pregnant CTL and insulin added to phosphorylate IRbeta. This will allow us to evaluate if the rise in TNF- alpha in late pregnancy induces serine phosphorylation of IRS-1 and converts IRS-1 to an inhibitor of IRbeta tyrosine kinase activity. Specific aim #3 will evaluate 24 women; 8 women (4 NGT and 4GDM) who do not plan to conceive (at yearly intervals), and 16 women (8 NGT and 8 GDM) planning to conceive (prior to conception, at 30-36 weeks and post partum. All subjects will be evaluated using densitometry, an intravenous glucose tolerance test, indirect calorimetry and the euglycemic clamp with infusion of [6-6/2H2] glucose. These data will allow us to evaluate the effect of pregnancy on pancreatic beta cell function and insulin sensitivity in women with NGT and GDM, in contrast to changes in thee measurements ascribed to time alone. The information obtained from these studies will allow us to begin to understand the mechanisms involved in the alterations in insulin sensitivity during pregnancy, and the potential genetic factors responsible for decreased glucose insulin sensitivity in women with GDM.